The human endometrium is essential in providing the site for implantation and maintaining the growth and survival of the conceptus. An unreceptive endometrium and disrupted maternal−conceptus ...interactions can cause infertility due to pregnancy loss or later pregnancy complications. Despite this, the role of uterine glands in first trimester human pregnancy is little understood. An established organoid protocol was used to generate and comprehensively analyze 3-dimensional endometrial epithelial organoid (EEO) cultures from human endometrial biopsies. The derived EEO expand long-term, are genetically stable, and can be cryopreserved. Using endometrium from 2 different donors, EEO were derived and then treated with estrogen (E2) for 2 d or E2 and medroxyprogesterone acetate (MPA) for 6 d. EEO cells were positive for the gland marker, FOXA2, and exhibited appropriate hormonal regulation of steroid hormone receptor expression. Real-time qPCR and bulk RNA-sequencing analysis revealed effects of hormone treatment on gene expression that recapitulated changes in proliferative and secretory phase endometrium. Single-cell RNA sequencing analysis revealed that several different epithelial cell types are present in the EEO whose proportion and gene expression changed with hormone treatment. The EEO model serves as an important platform for studying the physiology and pathology of the human endometrium.
The endometrium is the inner lining of the uterus that undergoes complex regeneration and differentiation during the human menstrual cycle. The process of endometrial shedding, regeneration, and ...differentiation is driven by ovarian steroid hormones and prepares the endometrium and intrauterine environment for embryo implantation and pregnancy establishment. Endometrial glands and their secretions are essential for pregnancy establishment, and cross talk between the glandular epithelium and stromal cells appears vital for decidualization and placental development. Despite being crucial, the biology of the human endometrium during pregnancy establishment and most of pregnancy is incomplete, given the ethical and practical limitations of obtaining and studying endometrium from pregnant women. As such, in vitro models of the human endometrium are required to fill significant gaps in understanding endometrial biology. This review is focused on the evolution and development of in vitro three-dimensional models of the human endometrium and provides insight into the challenges and promises of those models to improve women's reproductive health. Summary sentence This review describes the evolution of in vitro models of human endometrium and potential applications for the study of endometrial function and dysfunction in women.
Characterization of the implanting human fetus as an allograft prompted a field of research in reproductive immunology that continues to fascinate and perplex scientists. Paternal- or partner-derived ...alloantigens are present in the maternal host at multiple times during the reproductive process. They begin with exposure to semen, continue through implantation and placentation, and may persist for decades in the form of fetal microchimerism. Changes in maternal immune responses that allow allogenic fertilization and survival of semiallogenic concepti to delivery must be balanced with a continued need to respond appropriately to pathogenic invaders, commensals, cell or tissue damage, and any tendency toward malignant transformation. This complex and sophisticated balancing act is essential for survival of mother, fetus, and the species itself. We will discuss concepts of alloimmune recognition, tolerance, and ignorance as they pertain to mammalian reproduction with a focus on human reproduction, maternal immune modulation, and the very earliest events in the reproductive process, fertilization and implantation.
Macrophages are versatile cells that play a central role in innate and adaptive immunity and participate in a wide variety of biological processes. In the uterine decidua, macrophages represent a ...major leukocyte subset throughout pregnancy. Here, decidual macrophages exert an immunosuppressive phenotype characterized by abundant production of interleukin (IL)-10 and indoleamine 2,3-dioxygenase activity. Their polarized cytokine secretion pattern has recently been classified as an M2 phenotype. These features of decidual macrophages favor maternal immune tolerance to semiallogenic fetus. In addition, macrophages cooperate with trophoblast cells during the early stages of human pregnancy to support uterine vasculature remodeling by removing apoptotic cells and through the production of proteases that degrade the extracellular matrix. In the peripartum period, macrophages also participate in the regulation of cervical ripening and the initiation of parturition through the production of proinflammatory cytokines and prostaglandin E(2) (PGE(2)). Aberrant activity of uterine macrophages is linked to the pathogenesis of preeclampsia and preterm delivery. Here, we review the immunomodulatory roles of decidual macrophages during pregnancy.
Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is ...most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to lowtiters (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.
Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 ...signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall.
Macrophages represent one of the major leukocyte subsets in the uterine decidua. Owing to their remarkable phenotypic plasticity, decidual macrophages can participate in diverse activities during ...pregnancy. At baseline, decidual macrophages are characterized by an immunosuppressive phenotype and M2 polarization, supporting feto-maternal immune tolerance. In early pregnancy, macrophage-derived pro-angiogenic factors prompt vascular remodeling within the uterine wall to ensure appropriate utero-placental circulation. Upon invasion by pathogens, pattern recognition receptors on decidual macrophages help to alter the characteristics of these malleable cells toward an M1, inflammatory phenotype. Similar inflammatory characteristics are seen in those macrophages that accumulate in the lower segment of the uterus to drive cervical ripening. Disturbances in the tight control that balances macrophage function during pregnancy can trigger the development of pregnancy complications. Here, we discuss the physiologic role of uterine macrophages at different stages of pregnancy and describe their relevance in selected pregnancy disorders.
The syncytialization of cytotrophoblast cells to syncytiotrophoblast is central to human placental transport and hormone production. Many techniques for in vitro study of this process have been ...proposed and new investigators to the field may find the literature in the field daunting. Here, we present a straightforward and reliable method to establish this important model using modern but readily available tools and reagents.
Villous cytotrophoblast cells are obtained from term placenta using mild enzymatic degradation, Percoll gradient centrifugation, negative magnetic cell sorting using an antibody against classical major histocompatibility complex molecules and in vitro culture on a matrix-coated growth surface.
The purity of isolated cytotrophoblast cells exceeds 98 % as assessed by cytokeratin-7 expression using flow cytometry. Contamination by mesenchymal cells, extravillous trophoblast cells, leukocytes, Hofbauer and endothelial cells is minimized (less than 2 % when analyzed for vimentin, HLA-G, CD45, CD163 and CD31 using flow cytometry). Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12 h of plating. By 72 h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular E-cadherin expression upon fusion into multinucleated syncytia. After 72 h in culture, nearly every cultured cell expresses syncytiotrophoblast markers, including cytokeratin-7, human chorionic gonadotropin-β (β-hCG) and the fusion-related proteins glial cell missing-1 (GCM-1) and syncytin.
We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.
To describe the natural history and outcomes of a large cohort of expectantly managed angular pregnancies diagnosed in the first trimester by specific ultrasound criteria.
We conducted a prospective ...case series of women with prenatally diagnosed angular pregnancy at a single academic tertiary care center from March 2017 to February 2019. Participants were identified at first-trimester ultrasound scan using specifically proposed diagnostic criteria for angular pregnancy and followed prospectively. Maternal and fetal data were gathered from the medical record.
Forty-two cases of angular pregnancy were identified at first-trimester ultrasound scan. At presentation, 33 patients (79%) were asymptomatic, eight (19%) had vaginal bleeding, and two (5%) had pain. The mean gestational age at diagnosis was 7.4±1.0 weeks; the mean myometrial thickness was 5.1±1.6 mm (95% CI 4.6-5.6). At initial follow-up about 2 weeks later, 23 patients (55%) had ultrasound scans that normalized, 13 (31%) cases persisted as angular pregnancies, and six (14%) resulted in early pregnancy loss. After each gestation had been followed until completion, 33 (80%) pregnancies resulted in live birth and eight (20%) in early pregnancy loss. One patient was lost to follow-up. Of the 33 live births, 24 (73%) were vaginal deliveries, nine (27%) were cesarean deliveries, 27 (82%) were term deliveries, and six (18%) were preterm deliveries. There were no cases of uterine rupture, maternal death, abnormal placentation, or hysterectomy.
In 42 cases of angular pregnancy diagnosed by first-trimester ultrasound examination, outcomes were largely positive, with an 80% live-birth rate and a 20% early pregnancy loss rate. Early diagnosis of angular pregnancy using the described criteria may represent an entity that more closely resembles a normal, noneccentric intrauterine pregnancy rather than an ectopic pregnancy. Therefore, most cases can be closely observed and efforts made to expectantly manage pregnancies while awaiting viability.
Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been ...challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7 ⁺ within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7 ⁺ and in which the majority of cells are transiently CDX2 ⁺. Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.
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